Effect of ultrasonic activation on calcium ion quantification, smear layer removal, and canal cleaning efficacy of demineralizing irrigants.

BACKGROUND: Effective removal of smear layer ensures better penetration of irrigants ensuring root canal disinfection and has the potential of sealing dentinal tubules during obturation. AIM: The aim of this study is to evaluate the canal cleaning efficacy and calcium ion liberation with the use of demineralizing irrigants, namely, 17% ethylenediaminetetraacetic acid (EDTA), 7% maleic acid, and 10% citric acid with or without ultrasonic (US) activation.
MATERIALS AND METHODS: Crowns of 62 extracted maxillary central incisors were decoronated and root canals prepared. Based on the final irrigant teeth were divided into following groups (n = 10), namely, 10% citric acid, 7% maleic acid, 17% EDTA, and 10% citric acid with US activation, 7% maleic acid with US activation, and 17% EDTA with US activation. Irrigants were subjected to inductively coupled plasma-atomic emission spectroscopy followed by debris scoring and scanning electron microscope analysis. STATISTICAL ANALYSIS: One-way analysis of variance with the Bonferroni's correction for multiple testing was employed.
RESULTS: About 10% citric acid with US activation liberated the highest calcium ions and removed the maximum smear layer.
CONCLUSIONS: Citric acid with US activation is a potentially effective irrigation regimen with sodium hypochlorite.

INTRODUCTION

The use of either hand or mechanized rotary and reciprocating endodontic instruments in the root canal space creates considerable amount of dentinal debris to form the smear layer. This smear layer consists of dentin shavings, cell debris, and pulp remnants and can be described as having two separate layers: a loose, superficial deposit and an attached stratum that extends into the dentinal tubules, forming occluding plugs. Identification of the smear layer was made possible using the electron microprobe with scanning electron microscope (SEM) attachment.

Use of chelating agents such as ethylenediaminetetraacetic acid (EDTA), acids-like citric acid, maleic acid, and the use of sonics, ultrasonics (USs) has been previously done to remove the smear layer. EDTA as a chelating agent combines with calcium ions in the tooth structure unlike acids such as citric acid, maleic acid, and tannic acid which dissolve inorganic structure as a function of their low pH. The efficacy of EDTA to remove smear layer has been widely investigated.[1234567891011121314] The chelating effectiveness of EDTA is limited as no additional dissolution of calcium ions from the tooth takes place after all the ions are bound, and equilibrium is reached. Alternatives to EDTA are sought as it is not naturally occurring and is considered a potential pollutant.[9] In addition, the application and volume of EDTA >1 min or 1 mL are known to cause dentinal erosion.[15] The high cytotoxicity of EDTA at concentrations indicated in endodontics, and relatively lower cytotoxicity of citric acid, have triggered the search for another demineralizing irrigant.

Citric acid is a weak organic acid that reacts rapidly with calcium ions. Smear layer removal by varying concentrations of citric acid including 10%, 15%, 20%, and 42% has been done previously.[678910] A recent study by Silva et al.[11] showed effective smear layer removal by 10% citric acid when compared to EDTA. However, there is no unified conclusion regarding the same across investigations.[7910]

Maleic acid has established etching in adhesive dentistry and smear layer removal in endodontics. Smear layer removing the ability of maleic acid has been comparable to EDTA[9] or even greater than it.[1216]

Passive US irrigation of final irrigants has shown greater smear layer removal than conventional syringe irrigation.[17] It is interesting however that majority of investigations analyzing smear layer removal by decalcifying agents have done so by flushing the irrigant in the root canal without US activation of the same.[126789101112] Studies by Lui et al.[18] and Schmidt et al.[19] have found that the use of USs with 17% EDTA improves smear layer removal. However, similar studies for maleic acid and citric acid are missing from endodontic literature to the best of our knowledge.

Therefore, the purpose of this study was two-fold; first, to quantify the calcium ions liberated by inductively coupled plasma–atomic emission spectroscopy (ICP-AES) of three different irrigating solutions, namely, 17% EDTA, 7% maleic acid, and 10% citric acid with and without the effect of US activation and second, to evaluate the canal cleaning efficacy of same by debris scoring.

The null hypothesis tested was that there is no difference in the calcium ion removing the ability of the tested demineralizing irrigants with and without US activation.

MATERIALS AND METHODS

Sixty-two freshly extracted single-rooted human maxillary central incisors were a part of the study and were disinfected by storing in 10% formalin for 2 weeks. Radiographs were made to confirm single roots and straight canals. Teeth were then ultrasonically cleaned and decoronated at the cementoenamel junction using a diamond disc with water spray. A size 10 K file (MANI, Inc., Tochigi, Japan) was introduced in the canal to maintain apical patency. After establishing working length step-back preparation of the root canals with K-type hand, instruments in 1 mm increments was done. After gauging, the master apical file size corresponded to an ISO size 40 and a final ISO file size of 80 was used in all the teeth specimens. Care was taken to keep the apex patent. The canal was irrigated with 1 mL of 3% sodium hypochlorite (NaOCl) (Prime Dental Products Pvt. Ltd, Thane, Maharashtra, India) between each instrument change and 5 mL of 3% NaOCl after complete instrumentation. Finally, the canal in each tooth was rinsed with 5 mL of deionized water.

Each tooth was placed in a 15 mL falcon tube with the lid perforated in such a way that the crown portion of the tooth remained outside the lid, and the root portion was inside the tube. The gap between the tooth and the perforation on the lid was sealed with Filtek Z250 composite resin (St. Paul, MN, USA). At this point, 60 of the 62 teeth positioned in their respective tubes were randomly divided into six groups of ten samples each. Each group represented a different test irrigant, namely, 10% citric acid, 7% maleic acid, 17% EDTA, 10% citric acid with US activation, 7% maleic acid with US activation, and 17% EDTA with US activation. The remaining two teeth were used as control and irrigated with distilled water. All the solutions were freshly prepared before utilizing in the research laboratory.

In the groups without US activation and the control group, 5 mL of the test irrigating solution was delivered inside the canal with a 27G beveled needle so that the irrigant flowed through the entire length of the canal and exited through the patent apical foramen into the collection tube below. To prevent backflow of the irrigant, Filtek Z250 light cure resin was used to seal the space between canal entrance and needle. In the groups with US activation, 1 mL of irrigant was delivered inside the root canal, and US activation was done using a size 15 K file fitted onto a Suprasson P-50 unit (Satelec, Merignac, Cedex, France). The file tip placed was placed 1-mm short of the working length and activated for 1 min at a power setting of two. A final flush was done with the remaining 4 mL of the test irrigating solution.

Inductively coupled plasma-atomic emission spectroscopy

After collection of the solution in the tubes, the lids were removed and the teeth separated from the lids. The tubes were labeled with identical new caps without perforations for analysis by ICP-AES. Ten tubes were used for each test solution to determine the calcium ion concentration and a mean value was calculated for each group. An ICP-AES (Arcos, MS Spectro, Germany) was used to carry out the procedure. EDTA was diluted using deionized water to prevent it from quenching the plasma flame of the spectrometer. The calcium ion concentration values thus obtained were tabulated.

Stereomicroscopy

Two diametrically opposite grooves were made in the teeth using diamond discs under cooling, and a bi-bevel chisel was used to split the teeth in half lengthwise. Teeth which showed evidence that the groove had penetrated inside the root canal or teeth which showed irregular cleavage were discarded and replaced with a new specimen. The hemisected side with fewer irregularities which best represented the entire length of the root canal from coronal to the apical third was used for microscopy. The hemisected teeth were examined under a stereomicroscope at ×40 for observing the debris present on the root canal walls. The debris scoring system given by Peters OA was used.[4] Scoring was done separately by two-blinded examiners. Assessment of scores for the first 20 samples was done by both the examiners together for calibration purposes. Intra- and inter-examiner reliability was verified by kappa test.

Scanning electron microscopy

A randomly chosen sample representative of each group was processed for SEM. The individual sections were mounted on aluminum stubs, desiccated, sputter coated with gold, and positioned in an SEM (EDAX, New Jersey, USA). Photomicrograph images were then captured by consensus of two examiners who chose multiple areas of the dentine wall that best represented each third of the whole specimen. Photomicrographs were captured at magnifications of ×1000 and ×2000 at three levels as follows: coronal, middle, and apical to see the presence or absence of smear layer. All images in this article are at ×2000 magnification.

The data for the calcium ion concentration and debris score were tabulated and subjected statistical analysis using SPSS software version 21 (SPSS Inc., Chicago, IL, USA) to one-way analysis of variance with Bonferroni's correction for multiple testing. A significance level of 5% was adopted.

RESULTS

Debris scoring

Kappa test values for with significance set at 0.5 was 0.9 and above for different groups. Table 1 shows mean debris scores for each group. No difference was observed in debris scores of middle and apical thirds between groups (P > 0.05). However in the coronal third, 7% maleic acid showed significantly higher debris than 10% citric acid and 17% EDTA with US (P < 0.05).

Table 1

Mean (standard deviation) values of debris in thirds of canal irrigated with different demineralizing agents

Calcium ion quantification

Table 2 presents the means and standard deviations of the calcium ion concentration of the test different solutions after final irrigation. Calcium ion concentration in citric acid group with US activation was significantly greater than all other demineralizing agents (P < 0.05). Statistical analysis revealed no significant difference between 7% maleic acid, 17% EDTA both with and without US activation and 10% citric acid without US activation. US activation resulted in significantly higher calcium ion concentration only with citric acid. In the other two irrigants, namely, maleic acid and EDTA US activation did not liberate higher calcium ions.

Table 2

Mean (standard deviation) of the calcium ion concentration of the different test solutions

Figure 1 shows photomicrographs of all groups for coronal, middle, and apical thirds.

Figure 1

Photomicrographs showing coronal, middle and apical thirds (rows) of 10% citric acid (a, h and o), 7% maleic acid (b, I and p), 17% ethylenediaminetetraacetic acid (c, j and q), 10% citric acid with ultrasonic (d, k and r), 7% maleic acid with ultrasonic (e, l and s), 17% ethylenediaminetetraacetic acid with ultrasonic (f, m and t) and control (g, n and u) respectively. Note the crystals in citric acid with ultrasonic denoted by arrows

Control

All thirds were smeared from end to end. Smooth amorphous smear layer obscured dentinal tubules with the presence of debris on the surface.

Citric acid

Coronal third displayed a smear free surface with almost all the tubules free from smear plugs. The middle third showed walls free from smear, but debris plugs present in some tubules. The apical third showed intact zones of smear layer and only a few visible tubule orifices.

Maleic acid

The coronal third showed some tubule orifices with incompletely removed smear layer. From middle to apical third the walls appeared increasingly covered by surface smear with debris plugs in the mouth of tubules. All thirds showed discrete clumps of debris.

Ethylenediaminetetraacetic acid

Walls of all thirds were evenly covered by surface smear.

Citric acid with ultrasonic

Coronal and middle thirds were characterized by the absence of smear layer and patent dentinal tubules. However, prominent crystals were evident in both thirds. Energy-dispersive X-ray analysis (EDXA) of the samples showing crystals to study the elemental composition showed carbon, oxygen, and calcium as major elements. Apical thirds were debris free and largely smear free, but tubule openings were not entirely open and showed partially intact smear plugs.

Maleic acid with ultrasonic

Smooth amorphous smear layer with uniformly scattered debris was observed in the coronal third. Middle third showed the absence of smear layer and partially patent tubule openings. The apical third showed the presence of smear layer in the majority of areas with patches of dentinal tubule openings occluded with smear plugs.

Ethylenediaminetetraacetic acid with ultrasonic

Coronal and middle third were characterized by a smear free surface and patent dentinal tubule openings. Apical third showed walls covered by smear layer with only a few patent tubules.

DISCUSSION

The following aspects could be inferred based on the literature for the methodology applied; several studies used SEM technique to investigate the ability of smear layer removal. A disadvantage of this method is that only an insignificant area of the root canal can be evaluated compared to the total root canal area,[20] and this area often is not standardized with a bias of SEM operator to select smear free dentin regions than smear-coated areas.[21] To address this debris issue scoring was done under ×40 magnification with a stereo microscope. A relationship of smear layer removal and the quantity of calcium ions present in the irrigating solutions after use has been already suggested.[5] Therefore, calcium ion quantification was done with SEM to validate the results. Thus, for a comprehensive understanding of debris removal, smear layer removal and calcium ion removal for each irrigant, investigative methods that determined aforementioned criteria be employed and findings correlated. As the SEM photomicrographs of the samples were following the findings of calcium ion quantification, it is safe to assume that greater calcium ion values represent better smear layer removal.

The amount of calcium ions present in a chelating agent has been quantified by atomic absorption spectroscopy (AAS) previously.[5911] In this study, however, ICP-AES was used as it is a more sensitive method than AAS. ICP generates a much higher temperature than does a flame; 6000–8000 K for ICP-AES versus 1700–2700 K for AAS. This higher temperature results in greater atomization and excitation efficiencies of elements. Hence, the detection limits are approximately 1 part per billion for ICP-AES vs. approximately 0.1 parts per million for flame AAS. The application field originally assigned to AAS, using both the flame and graphite furnace atomic absorption spectrometry, has been relinquished to the ICP.[22]

Although US activation resulted in greater calcium ion removal with citric acid (80.91 ± 31.9 vs. 10.09 ± 3.89), maleic acid (18.81 ± 8.91 vs. 3.07 ± 1.45), and EDTA (9.84 ± 2.69 vs. 4.26 ± 1.74) calcium ion removal by citric acid activation alone was statistically significant. The reasons for this finding could probably be attributed to the effects of acoustic microstreaming and cavitation. Factors such as the pH and surface tension of freshly prepared solutions used in the present study could have contributed toward greater calcium ion removal with 10% citric acid as compared 7% maleic acid and 17% EDTA, however, they were they outside the constraints of this investigation. Lower calcium ion liberation values of EDTA with US activation can probably be explained due to the rapidly reducing pH of EDTA due to chelation which makes it less effective with time.[23] Our results are in partial agreement with Ciucchi et al.[24] who concluded that US activation of EDTA did not result in greater smear free surfaces; however, the same was not found to be true with regards to US activation of citric acid. Both the ICP-AES and SEM findings showed that 10% citric acid resulted in highest calcium ion and smear layer removal among all demineralizing agents. However, the debris scores did not corroborate with these findings. This indicates that although demineralizing agents facilitate smear layer removal, they do not possess debris removing ability and thus cannot compensate for inadequately debrided canals.

The present study found no difference in calcium ion concentration of demineralizing irrigants without US activation and supports the SEM findings of Khedmat and Shokouhinejad[7] and Wu et al.[10] These results were neither in agreement with Spano et al.[9] nor Silva et al.[11] who found 10% citric acid and 15% EDTA, respectively, to yield greater calcium ions. Similarly, our results disagree with Kumar et al.[25] who found 10% citric acid to cause greater demineralization compared to EDTA.

It is noteworthy that SEM photomicrographs for 10% citric acid with USs revealed precipitation of crystals on the canal wall, especially in the coronal and middle thirds. Similar crystals were observed by Yamada et al.[1] on using 25% citric acid and could not explain their formation. In some instances, photomicrographs showed end-to-end crystal deposition, thus covering vast areas of dentin walls and questioning the ability of citric acid to maintain the patency of dentinal tubules. Although EDXA showed carbon, oxygen and calcium as major elements of these crystals, whether these crystals can be removed by a final rinse of sterile water was beyond the scope of the present study.

Although our results did not exactly replicate the SEM findings of Lui et al.[18] and Schmidt et al.[19] who found that US activation of EDTA improved smear layer removal, they seem to suggest a definite correlation between calcium ion removal and US activation of demineralizing irrigants. The present study showed that maleic acid with or without US activation did not liberate higher calcium ion removal. These findings are contrary to those of Ulusoy and Görgül[12] and Kuruvilla et al.[16] In spite of using lesser quantity of irrigant (1 mL vs. 5 mL) found better smear layer removal. This could probably be the result of longer time used for irrigation and the order of irrigants used. It is noteworthy that while the order of irrigants used in our study was NaOCl followed by maleic acids, Ulusoy and Görgül[12] used the same irrigants in reverse order.

As the purpose of our study was to investigate the Ca ion removal by demineralizing irrigants, a final NaOCl rinse was not done. A NaOCl rinse would have probably resulted in greater smear plug removals and patent dentinal tubule openings. Considering the limitations and based on the findings of this in vitro study the null hypothesis was rejected.

CONCLUSIONS

It clearly shows that 10% citric acid with US activation liberated the highest calcium ions and removed the highest smear layer among all irrigants. There was no difference in calcium ion liberation capacity all other irrigants as well as in the canal cleaning efficacy between various groups. Using citric acid with US activation followed by a final rinse with sterile water to remove the crystals could be a good irrigation regimen with sodium hypochlorite.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.